These studies are directed toward the investigation of the structural basis of the interaction of PP1 with two inhibitory proteins, inhibitor- 2 and inhibitor-3. Inhibitor-2 is unique among PP1 inhibitors in that it is able to interconvert PP1 between active and inactive conformations that are stable when removed from I-2. The specific aims are directed toward identifying the binding sites of PP1 for inhibitor-2 and the determination of the role of metal ions in the interconversion process. A hypothesis that the mechanism for the activation/inactivation of PP1 by inhibitor-2 involves changes in the metal site occupancy of PP1 will be tested by the use of 57/Co and a novel form of recombinant PP1 (PP1/Co). Mutational analysis will be tested to characterize three putative sites on PP1 for the binding of inhibitor-2. Analysis of the mutants will involve examination of the inhibition, inactivation and activation reactions of I-2, to determine if there is a site specific relationship with these individual effects of I-2. Inhibitor-3 is a noel PP1 inhibitor protein, and the studies are directed toward the identification of the sites on I-3 that are responsible for binding to PP1. The question of whether occupancy of the VxF-binding site is sufficient to elicit functional changes in PP1 in the form of inhibitory effects or changes in substrate recognition will also be addressed. The crystallization of PP1/Co, a novel active conformer of PP1, and its complex with inhibitor-3 will be pursued in order to allow the determination of their crystal structure in collaboration with a crystallography laboratory. The regulation and potential cellular roles of I-3 will be investigated. The inactivation of the inhibitory activity of inhibitor-3 by phosphorylation will be studied, in order to understand how it functions a regulator of PP1. The phosphorylation sites for PKA, CK2 and PKC, and their effects on the inhibitory properties of inhibitor-3 will be examined. In order to understand the potential roles of inhibitor-3, the following will be determined: i) its subcellular localization, ii) whether it functions as a targeting protein, iii) if its is rapidly turned over, and iv) it is phosphorylated in vivo. The possible variation of these properties during the cell cycle will also be investigated. An expression system in mammalian cells will be established to investigated the phenotypic effects of over-expression of I-3, and to determine if mutants which are defective in binding to PP1 behave as dominant negative mutants. In order to obtain insights into the potential functions of I-3, the functions of the cognitive yeast gene YFR003c will be investigated.